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To understand the allosteric modulation dynamics of non-nucleoside reverse transcriptase inhibitors (NNRTIs), various models and suggestions have been derived from crystallography and simulation. Here, using a new force field, ff12SB, and GPU parallel computing technology, we performed 100-ns-long molecular dynamics simulations on three reverse transcriptase (RT) systems, one bound to inhibitor Efavirenz (EFV) and the others free. Analyses of the influence of the EFV on the conformation of the RT, flexibility of residues and dynamic behaviors of the systems were conducted. The simulations indicate that EFV binding induces structural distortion of the RT, whereas the configuration of the RT is more stable during dynamics, along with a decreasing extent of motion of the residues. EFV suppresses the flexibility of the thumb subunit and reduces that of most residues in the fingers subdomain as well, suggesting that EFV causes not only the so-called“thumb arthritis” but also a slight“fingers arthritis”. No conformational transition occurred throughout the entire simulations and the samples maintained their starting conformations, i.e., free RT with a closed conformation stayed in the functional state and EFV-bound RT remained in open conformation. However, EFV-free RT with an initially open conformation exhibited an evident trend toward the closed state. These results agree with the models from experiments, and present a useful insight into the allosteric inhibition mechanism of NNRTIs. In addition, the simulation methodology has been discussed in detail and will be of significance to the computational simulation of large biological molecules. 436 孟现美等:分子动力学模拟别构抑制剂Efavirenz对HIV-1逆转录酶的作用 No.2 1 引 言 逆转录酶(RT)在 I型人体免疫缺陷病毒(HIV-1) 复制过程中,可以催化病毒RNA基因组使之逆转 录为双链DNA病毒前体,因此是发明抗艾滋病 (anti-AIDS)新药的一个重要靶酶 。目前,以RT为 靶治疗艾滋病的药物中,非核苷类 RT抑制剂 (NNRTIs)被认为是一类具有前景的特效药 2 。 NNRTIs也称为别构抑制剂,结合在RT的非活性 位,可以避免活性位点或底物的选择性压力,具 有广谱抗HIV-1耐药性的特点。如图 1A所示,RT 是一个由 p66和 p51两个亚基组成的异二聚体 。 p66亚基由DNA聚合酶和RNase H结构域构成,其 中,DNA聚合酶结构域形状类似于握杯的右手 形,它的 3个子域形象地称之为手指(fingers)、手 掌(palm)和拇指(thumb)子域,并通过所谓的连接子 (connection)与RNase H结构域连接在一起。p51亚 基具有与p66聚合酶结构域同样的氨基酸序列,但 折叠方式不同。如图 1B所示,在 p66的拇指和手 掌连接处有一个疏水性口袋结构 (NNRTIBP), NNRTIs可以进入并束缚在袋内 。图中显示的EFV (Efavirenz)属于第二代NNRTIs类药物。 然而,关于NNRTIs和RT的作用机制当前还 无一致的理解。一般观点是建立在实验晶体数据 分析基础上的“拇指关节炎”或“分子楔”模 型 -,但最近的荧光实验却意味NNRTIs的作用会 使手指-拇指“握度松动(grip loosening)”,从而抑 制聚合催化过程 。理论上也相应地开展了许多工 作 -,主要是利用分子动力学方法研究具有不同 初始结构和不同底物的RT动力学性质。由于RT 体系过大,所以模拟时间一般只有几个纳秒,所 得计算结果也不同。例如,文献 11(3 ns)和 12(2.5 ns)的模拟结果支持“拇指关节炎”模型;文献 13 (1 ns)模拟结果表明,去掉NNRTIs后拇指子域会 迅速从张开塌向闭合状态转变;文献 14(1.1 ns)模 拟结果表明,底物会增加RT的柔性。近来,两个 较长时间的动力学模拟结果更是引人关注:Ivetac 和McCammon利用GROMACS程序和GROMOS 力场,对具有不同原子初速度的体系副本进行了 30 ns的模拟,发现无论APO(无任何抑制剂)还是 结合NNRTI的RT体系,其构象发生转变的几率都 相当可观(分别为 50%和 25%);Wright等 用AMBER9和 ff03力场进行的计算(模拟时间达 100 ns, 但步长较大为4 fs)发现,被别构抑制剂撑开的拇指 子域可以重返类似APO的闭合状态。显然,这些 模拟结果有悖于一般的“拇指关节炎”观点,意 味着RT的构象变化和NNRTI的作用机制可能并不 像一般认为的那样。换句话说,无论APO还是加 上NNRTI后,RT体系的能量曲面上都可能存在两 |
